Procalcitonin for the prognosis of adverse events

ABSTRACT

The present invention relates to an in vitro method for the prognosis of an adverse event in asymptomatic subjects comprising the determination of the level of Procalcitonin (PCT) or a fragment thereof or a precursor or fragment thereof having at least 12 amino acid residues in a sample of a bodily fluid from said subject and the correlation of the determined level to a potential risk of sustaining an adverse event.

FIELD OF THE INVENTION

The present invention is in the field of clinical diagnostics.Particularly the present invention relates to the determination of thelevel of Procalcitonin (PCT) in a sample derived from a bodily fluid ofa subject.

BACKGROUND OF THE INVENTION

Procalcitonin (PCT) is known as a biomarker, that reflects the presenceand severity of local and systemic bacterial infections, i.e. sepsis(Assicot et al., Lancet 1993; 341:515-8; Muller et al., Crit Care Med2000; 28:977-83.; Harbarth et al., Am J Respir Crit Care Med 2001;164:396-402; Becker et al., Crit Care Med 2008; 36:941-52; Becker etal., J Clin Endocrinol Metab 2004; 89:1512-25; Nobre et al., Am J RespirCrit Care Med 2008; 177:498-505; Christ-Crain et al., Lancet 2004;363:600-7; Stolz et al., Chest 2007; 131:9-19; Christ-Crain et al., Am JRespir Crit Care Med 2006; 174:84-93; Briel et al., Arch Intern Med2008; 168:2000-7; discussion 7-8).

During bacterial infections, plasma PCT concentrations are typicallyabove 0.25 ng/mL. More recently, using highly sensitive assaytechnologies, it has been found that in several non-infectious diseasesPCT concentrations can be elevated to the upper concentration range ofthe normal range or above, but below the concentrations which have beenknown to be associated with bacterial infections requiring antibacterialtreatment. Moreover, it has been found that these PCT concentrations areassociated with an unfavorable prognosis for these patients.

These diseases include coronary artery disease and acute coronarysyndromes. There are only two publications which describe thequantitative PCT concentrations in healthy individuals (Morgenthaler etal., Clin Chem 2002, 48:788-790; Morgenthaler et al., Clin Lab 2002,48:263-270). However, it is unknown whether relatively elevated PCTconcentrations in healthy individuals can be associated with theoccurrence of potential future adverse events. The possibility ofpredicting future adverse events for individuals which are apparentlyhealthy at presentation is important, since early recognition of risk isa prerequisite for initiating measures helping to prevent thedevelopment of adverse events.

It has thus been an object of the present invention to establish a linkbetween PCT levels in the apparently healthy (asymptomatic) populationwith the prognosis of adverse events.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that the PCTlevels in samples from asymptomatic subjects, who have later sufferedfrom an adverse event, are increased relative to PCT levels in samplesfrom asymptomatic subjects who will not suffer from an adverse event ina statistically relevant manner.

The present invention relates to an in vitro method for the prognosis ofan asymptomatic subject to experience an adverse event. The methodcomprises: First, the level of procalcitonin (PCT) or fragments thereofin a sample from the subject is determined. Secondly, the level ofprocalcitonin or fragments thereof is correlated to a risk of thesubject to suffer from an adverse event.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for determining the risk of anapparently healthy subject for suffering from an adverse event or fordetermining the presence of absence of an increased risk of anapparently healthy subject for suffering from an adverse event in thefuture. Preferably, the risk of the occurrence of adverse events duringa time period of about 6 months to 10 years can be predicted. The methodcomprises the following steps: First, a sample is provided from thesubject. Secondly, the level of procalcitonin (PCT) or of a fragmentthereof is determined in the sample. Thirdly, the risk of the subjectfor getting an adverse event based is determined based on the PCT levelor it is determined whether the subject has an increased risk forsuffering from an adverse event based on said PCT level.

The term “risk” refers to a probability for a subject to suffer in thefuture from a certain adverse event.

“Subject” as used herein refers to a living human or non-human organism.In a preferred embodiment, the subject is a human being. Furthermore, itis preferred that the subject is free of (symptomatic) bacterialinfections at the time the sample from the subject is obtained from thesubject. This ensures that the PCT levels are not increased above thenormal range in the subject. Therefore, the subject is preferably testedfor bacterial infections prior to performing the method of theinvention.

The “apparently healthy” subject is preferably also free of anynon-infectious diseases, i.e. the subject does not show any symptomsthat would allow a diagnosis of a non-infectious disease. The term“non-infectious disease” refers to diseases such as hypertension,orthopaedic diseases such as osteoporosis, and neurodegenerativediseases such as Alzheimer's disease. It is known that the PCTconcentration is enhanced in neonates because of the post-natal trauma.Therefore, neonates are excluded from the claims. Neonates according tothe definitions of the present inventions have preferably an age between1 day to 10 days, more preferably one day to 3 days. It is also knownthat PCT concentrations are enhanced in patients after surgery, e.g.after organ transplantation as heart transplantation, lungtransplantation, liver transplantation, kidney transplantation, cardiacsurgery. Thus, patients after after surgery, e.g. after organtransplantation as heart transplantation, lung transplantation, livertransplantation, kidney transplantation, cardiac surgery are excludedfrom the group of healthy persons if the surgery has been conductedwithin the last seven days, preferably post-surgery patients areexcluded if the surgery has been conducted within the last fourteendays. In another embodiment post-surgery patients are excluded whichunderwent surgery within the last two months. Preferentially,post-trauma patients and persons are excluded which experienced saidtrauma within the last 14 days, preferably 7 days.

The term “sample” as used herein refers to a sample of bodily fluidobtained for the purpose of diagnosis, prognosis, or evaluation of asubject of interest, such as a patient. Preferred test samples includeblood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, andpleural effusions. In addition, one of skill in the art would realizethat some test samples would be more readily analyzed following afractionation or purification procedure, for example, separation ofwhole blood into serum or plasma components.

Thus in a preferred embodiment of the invention, the sample is selectedfrom the group consisting of a blood sample, a serum sample, a plasmasample, a cerebrospinal fluid sample, a saliva sample and an urinesample or an extract of any of the aforementioned samples. Preferably,the sample is a blood sample, most preferably a serum sample or a plasmasample. In the present invention, the term “prognosis” denotes aprediction of how a subject's (e.g. a patient's) medical condition willprogress. This may include an estimation of the chance of an adverseevent to occur for said subject.

An “adverse event” or adverse outcome is defined as an event or outcomecompromising the health of an individual. Said adverse event is notrestricted to, but may be selected from the group comprising a cardiacevent, a cardiovascular event, a cerebrovascular event, a cancer,diabetes, and death due to all causes. An adverse event includesinfections, serious infections and sepsis-like systemic infections andsepsis. An adverse is not an event caused by an acute exogene inducedadverse event and/or exogene induced trauma. Exogene induced traumainclude those which may be induced by accidents, e.g. car accidents.Exogene induces trauma are therefore excluded from the group of adverseevents.

A cardiac event was defined as the sum of any of the following events:acute myocardial infarction (ICD-code 410), acute and subacute ischemicheart disease (ICD-code 411), and the following procedures: coronaryartery bypass grafting or percutaneous transluminal coronaryangioplasty.

A cerebrovascular event was defined as the sum of any of the followingevents: occlusion or stenosis of the precerebral (ICD-code 433) orcerebral arteries (ICD-code 434).

Cardiovascular mortality was defined as death due to a cardiac orcerebrovascular event. Total cardiovascular events were defined as thesum of cardiac events, cerebrovascular events and cardiovascularmortality.

As mentioned herein, in the context of proteins or peptides, the term“fragment” refers to smaller proteins or peptides derivable from largerproteins or peptides, which hence comprise a partial sequence of thelarger protein or peptide. Said fragments are derivable from the largerproteins or peptides by saponification of one or more of its peptidebonds.

Procalcitonin in the context of the present invention preferably relatesto a peptide spanning amino acid residues 1-116, 2-116, or 3-116 orfragments thereof. Thus, the length of procalcitonin fragments should beat least 12 or 15 amino acids, preferably more than 50 amino acids, morepreferably more than 100 or 110 amino acids.

PCT may comprise posttranslational modifications such as glycosylation,liposidation or derivatisation. PCT itself is a precursor of calcitoninand katacalcin. The amino acid sequence of PCT 1-116 is given in SEQ IDNO. 1.

The term “level” in the context of the present invention relates to theconcentration (preferably expressed as weight/volume; w/v) of PCT (or afragment/precursor) in a sample taken from a subject.

Determining (or measuring or detecting) the level of PCT or a fragmentor a precursor or fragment thereof herein is performed using a detectionmethod and/or a diagnostic assay as explained below.

As mentioned herein, an “assay” or “diagnostic assay” can be of any typeapplied in the field of diagnostics. Such an assay may be based on thebinding of an analyte to be detected to one or more capture probes witha certain affinity. Concerning the interaction between capture moleculesand target molecules or molecules of interest, the affinity constant ispreferably greater than 10⁸ M⁻¹.

In the context of the present invention, “capture molecules” aremolecules which may be used to bind target molecules or molecules ofinterest, i.e. analytes (i.e. in the context of the present inventionthe cardiovascular peptide(s)), from a sample. Capture molecules mustthus be shaped adequately, both spatially and in terms of surfacefeatures, such as surface charge, hydrophobicity, hydrophilicity,presence or absence of Lewis donors and/or acceptors, to specificallybind the target molecules or molecules of interest. Hereby, the bindingmay for instance be mediated by ionic, van-der-Waals, pi-pi, sigma-pi,hydrophobic or hydrogen bond interactions or a combination of two ormore of the aforementioned interactions between the capture moleculesand the target molecules or molecules of interest. In the context of thepresent invention, capture molecules may for instance be selected fromthe group comprising a nucleic acid molecule, a carbohydrate molecule, aRNA molecule, a protein, an antibody, a peptide or a glycoprotein.Preferably, the capture molecules are antibodies, including fragmentsthereof with sufficient affinity to a target or molecule of interest,and including recombinant antibodies or recombinant antibody fragments,as well as chemically and/or biochemically modified derivatives of saidantibodies or fragments derived from the variant chain with a length ofat least 12 amino acids thereof.

The preferred detection methods comprise immunoassays in various formatssuch as for instance radioimmunoassay (RIA), chemiluminescence- andfluorescence-immunoassays, Enzyme-linked immunoassays (ELISA),Luminex-based bead arrays, protein microarray assays, and rapid testformats such as for instance immunochromatographic strip tests.

The determination of the PCT level is preferably performed using a(polyclonal or preferably monoclonal) antibody, particularly an antibodythat specifically binds to the katacalcin moiety or the calcitoninmoiety of PCT.

The assays can be homogenous or heterogeneous assays, competitive andnon-competitive assays. In a particularly preferred embodiment, theassay is in the form of a sandwich assay, which is a non-competitiveimmunoassay, wherein the molecule to be detected and/or quantified isbound to a first antibody and to a second antibody. The first antibodymay be bound to a solid phase, e.g. a bead, a surface of a well or othercontainer, a chip or a strip, and the second antibody is an antibodywhich is labeled, e.g. with a dye, with a radioisotope, or a reactive orcatalytically active moiety. The amount of labeled antibody bound to theanalyte is then measured by an appropriate method. The generalcomposition and procedures involved with “sandwich assays” arewell-established and known to the skilled person (The ImmunoassayHandbook, Ed. David Wild, Elsevier LTD, Oxford; 3rd ed. (May 2005),ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. 2006February; 10(1):4-10. PMID: 16376134, incorporated herein by reference).

In a particularly preferred embodiment, the assay comprises two capturemolecules, preferably antibodies which are both present as dispersionsin a liquid reaction mixture, wherein a first labelling component isattached to the first capture molecule, wherein said first labellingcomponent is part of a labelling system based on fluorescence- orchemiluminescence-quenching or amplification, and a second labellingcomponent of said marking system is attached to the second capturemolecule, so that upon binding of both capture molecules to the analytea measurable signal is generated that allows for the detection of theformed sandwich complexes in the solution comprising the sample.

Even more preferred, said labeling system comprises rare earth cryptatesor rare earth chelates in combination with fluorescence dye orchemiluminescence dye, in particular a dye of the cyanine type.

In the context of the present invention, fluorescence based assayscomprise the use of dyes, which may for instance be selected from thegroup comprising FAM (5- or 6-carboxyfluorescein), VIC, NED,Fluorescein, Fluoresceinisothiocyanate (FITC), IRD-700/800, Cyaninedyes, such as CY3, CY5, CY3.5, CY5.5, Cy7, Xanthen,6-Carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), TET,6-Carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE),N,N,N′,N′-Tetramethyl-6-carboxyrhodamine (TAMRA), 6-Carboxy-X-rhodamine(ROX), 5-Carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6),Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes,such as BODIPY TMR, Oregon Green, Coumarines such as Umbelliferone,Benzimides, such as Hoechst 33258; Phenanthridines, such as Texas Red,Yakima Yellow, Alexa Fluor, PET, Ethidiumbromide, Acridinium dyes,Carbazol dyes, Phenoxazine dyes, Porphyrine dyes, Polymethin dyes, andthe like. In the context of the present invention, chemiluminescencebased assays comprise the use of dyes, based on the physical principlesdescribed for chemiluminescent materials in Kirk-Othmer, Encyclopedia ofchemical technology, 4^(th) ed., executive editor, J. I. Kroschwitz;editor, M. Howe-Grant, John Wiley & Sons, 1993, vol. 15, p. 518-562,incorporated herein by reference, including citations on pages 551-562.Preferred chemiluminescent dyes are acridinium esters.

In the method of the invention, several ways of determining an increasedrisk of an apparently healthy subject for suffering from an adverseevent can be applied. The method may involve comparing the level of PCTin the subject with a predetermined value for PCT in a comparablepopulation. This can be a single cut-off value, e.g. a median or mean orthe 75^(th), 90^(th), 95^(th) or 99^(th) percentile of a population.Alternatively, a range can be used, e.g. where the tested population isdivided equally or unequally into groups (e.g. a low, medium or highrisk group) or into quartiles, the lowest quartile being individualswith the lowest risk and the highest quartile being individuals with thehighest risk.

The predetermined value can vary among particular populations selected,depending on certain factors, such as gender, age, genetics, habits,ethnicity or alike.

Preferably, the risk determination is performed by using of thefollowing alternatives:

-   -   comparing the determined PCT or PCT fragment level with the        median of the level of PCT or fragments thereof in an ensemble        of predetermined samples from a population of apparently healthy        subjects, or    -   comparing the determined PCT or PCT fragment level with a        quantile of the level of PCT or fragments thereof in an ensemble        of predetermined samples from a population of apparently healthy        subjects, or    -   performing calculations based on Cox Proportional Hazards        analysis or on risk index calculations such as the Net        Reclassification Index (NRI) or the Integrated Discrimination        Index (IDI).

In other embodiments, odds ratio, or hazard ratio is used as a measureof a test's ability to predict risk or diagnose a disease.

In the case of an odds ratio, a value of 1 indicates that a positiveresult is equally likely among subjects in both the “diseased” and“control” groups; a value greater than 1 indicates that a positiveresult is more likely in the diseased group; and a value less than 1indicates that a positive result is more likely in the control group. Incertain preferred embodiments, markers and/or marker panels arepreferably selected to exhibit an odds ratio of at least about 1.5 ormore or about 0.75 or less, more preferably at least about 2 or more orabout 0.5 or less, still more preferably at least about 3 or more orabout 0.33 or less, even more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less. The term “about” in this context refers to +/−5% of agiven measurement.

In the case of a hazard ratio, a value of 1 indicates that the relativerisk of an endpoint (e.g., death) is equal in both the “diseased” and“control” groups; a value greater than 1 indicates that the risk isgreater in the diseased group; and a value less than 1 indicates thatthe risk is greater in the control group. In certain preferredembodiments, markers and/or marker panels are preferably selected toexhibit a hazard ratio of at least about 1.1 or more or about 0.91 orless, more preferably at least about 1.25 or more or about 0.8 or less,still more preferably at least about 1.5 or more or about 0.67 or less,even more preferably at least about 2 or more or about 0.5 or less, andmost preferably at least about 2.5 or more or about 0.4 or less. Theterm “about” in this context refers to +/−5% of a given measurement.

As can be seen e.g. in table 6, the inventors were able to establishcut-off values for PCT levels that are indicative for the subject tosuffer from a certain adverse event in the future. Generally, a subjectcan be determined of being at risk for suffering from an adverse eventwhen the PCT level in a serum sample is at least 0.015 ng/ml serum,preferably at least 0.02 ng/ml.

Specifically, the subject is determined of being at an increased riskfor suffering from a cardiac event or a cardiovascular event when saidPCT level in a serum sample is at least 0.0155 ng/ml serum. The subjectis determined of being at an increased risk for suffering from acerebrovascular event or from dying due to cardiovascular cause when thedetermined PCT level in a serum sample is at least 0.0215 ng/ml. Thesubject is determined of being at an increased risk for dying fromcancer when the PCT level in a serum sample is at least 0.0155 ng/ml.The subject is determined of being at an increased risk for dying fromcancer when the PCT level in a serum sample is at least 0.0155 ng/ml.The subject is determined of being at an increased risk for dying whensaid PCT level in a serum sample is at least 0.0205 ng/ml., and isdetermined of being at risk for suffering from diabetes when the PCTlevel in a serum sample is at least 0.0185 ng/ml.

It is preferred that in the method of the invention at least one furtherparameter is determined and used to determine the risk or increased riskof suffering from an adverse event. Such a further parameter can be aclinical parameter, a laboratory (i.e. biochemical) parameter and/or agenetic parameter.

Suitable threshold levels for the stratification of subjects intodifferent groups (categories) can e.g. be done by grouping a referencepopulation of patients according to their level of PCT into certainquantiles, e.g. tertiles, quartiles, quintiles or even according tosuitable percentiles. For each of the quantiles or groups above andbelow certain percentiles, hazard ratios can be calculated comparing therisk for an e.g. adverse outcome, i.e. an “unfavourable effect” orincreased risk, e.g. in terms of survival rate. In such a scenario, ahazard ratio (HR) above 1 indicates a higher risk for an adverse outcomefor the patients. A HR below 1 indicates a lower risk. A HR around 1(e.g. +/−0.1) indicates no elevated risk for the particular group ofpatients. By comparison of the HR between certain quantiles of patientswith each other and with the HR of the overall population of patients,it is possible to identify those quantiles of patients who have anelevated level and enhanced risk and thereby stratify subjects accordingto the present invention.

The sensitivity and specificity of a diagnostic and/or prognostic testdepends on more than just the analytical “quality” of the test, theyalso depend on the definition of what constitutes an abnormal result. Inpractice, Receiver Operating Characteristic curves (ROC curves), aretypically calculated by plotting the value of a variable versus itsrelative frequency in “normal” (i.e. apparently healthy) and “disease”populations (i.e. patients suffering from bacterial infections). For anyparticular marker, a distribution of marker levels for subjects with andwithout a disease will likely overlap. Under such conditions, a testdoes not absolutely distinguish normal from disease or patients withenhanced risk from those which have no enhanced risk with 100% accuracy,and the area of overlap indicates where the test cannot distinguishnormal from disease; enhanced risk from those who haven't. A thresholdis selected, above which (or below which, depending on how a markerchanges with the disease) the test is considered to be abnormal andbelow which the test is considered to be normal. The area under the ROCcurve is a measure of the probability that the perceived measurementwill allow correct identification of a condition. ROC curves can be usedeven when test results don't necessarily give an accurate number. Aslong as one can rank results, one can create a ROC curve. For example,results of a test on “disease” samples might be ranked according todegree (e.g. 1=low, 2=normal, and 3=high). This ranking can becorrelated to results in the “normal” population, and a ROC curvecreated. These methods are well known in the art (Hanley et al. 1982.Radiology 143: 29-36). Preferably, a threshold is selected to provide aROC curve area of greater than about 0.5, more preferably greater thanabout 0.7, still more preferably greater than about 0.8, even morepreferably greater than about 0.85, and most preferably greater thanabout 0.9. The term “about” in this context refers to +/−5% of a givenmeasurement.

The horizontal axis of the ROC curve represents (1-specificity), whichincreases with the rate of false positives. The vertical axis of thecurve represents sensitivity, which increases with the rate of truepositives. Thus, for a particular cut-off selected, the value of(1-specificity) may be determined, and a corresponding sensitivity maybe obtained. The area under the ROC curve is a measure of theprobability that the measured marker level will allow correctidentification of a disease or condition. Thus, the area under the ROCcurve can be used to determine the effectiveness of the test.

The odds ratio is a measure of effect size, describing the strength ofassociation or non-independence between two binary data values (e.g. theratio of the odds of an event occurring in test negative group to theodds of it occurring in the test positive group).

The positive predictive value (PPV), or precision rate, is theproportion of patients with positive test results who are correctlydiagnosed and/or predicted. It reflects the probability that a positivetest reflects the underlying condition being tested for. The negativepredictive value (NPV) is the proportion of patients with negative testresults who are correctly diagnosed and/or predicted. The total accuracyis the percentage of all patients classified correctly with the test.Specifically, the at least one clinical parameter can be selected fromthe group consisting of gender (i.e. male or female), age, systolicblood pressure, systolic blood pressure, diastolic blood pressure, bodymass index, waist circumference, waist-hip ratio, and smoking habit.

In particular, because PCT levels are significantly different betweenmale and female (see e.g. Table 2), the gender of the subject is aparameter that needs to be considered when performing the method of theinvention.

The at least one laboratory parameter can be selected from the groupconsisting of fasting blood or plasma glucose concentration,triglycerides, cholesterol concentration, and HDL cholesterolconcentration and subfractions thereof, LDL cholesterol concentrationand subfractions thereof, cystatin C, insulin, CRP, natriuretic peptidesof the A- and B-type as well as their precursors and fragments thereofincluding ANP, proANP, NT-proANP, MR-proANP, BNP, proBNP, NT-proBNP,GDF15, ST2, procalcitonin and fragments thereof, pro-adrenomedullin andfragments thereof including ADM, PAMP, MR-proADM, CT-proADM,pro-Endothelin-1 and fragments thereof including CT-proET-1, NT-proET-1,big-Endothelin-1, and Endothelin-1.

The at least one genetic parameter is preferably selected from the groupconsisting of single nucleotide polymorphisms (SNPs), and mutations.

The method of the invention provides information as to a possibleincreased risk of a subject for suffering from an adverse event, asdescribed above and herein. Based on this information, preventivemeasures can be taken in order to avoid the adverse event from occurringin the subject. The preventive measures may be, e.g. taking appropriatemedicine, following dietary restrictions, exercising, obtaining medicalsupervision and others.

In a further aspect, the invention refers to the use of a method asdescribed above and herein for determining an increased risk of anapparently healthy subject for suffering from an adverse event.

Furthermore, the invention pertains to the use of a kit comprising oneor more antibodies directed against PCT or a fragment thereof or aprecursor or fragment thereof for the prognosis of an asymptomaticsubject to experience an adverse event, i.e. for performing a method asdescribed herein.

EXAMPLES Study Population and Design

The study population was obtained from the Prevention of Renal andVascular Endstage Disease (PREVEND) study. The PREVEND study wasdesigned to investigate prospectively the natural course of albuminuriaand its relation to renal and cardiovascular disease in a large cohortdrawn from the general population (age ranged between 28 and 75 years)of the city of Groningen, the Netherlands. Details of the study design,recruitment, and procedures have been published elsewhere (Hiliege etal., Circulation 2002, 106:1777-1782; Hillege et al., J Intern Med 2001,249:519-526; Mahmoodi et al., JAMA 2009, 301:1790-1797). The PREVENDstudy was approved by the local medical ethics committee, UniversityMedical Center Groningen, and conformed to the principles outlined inthe Declaration of Helsinki. All participants gave written informedconsent.

Measurements

The participants underwent two outpatient visits to assess demographic,anthropometric, and cardiovascular risk factors. They completed aquestionnaire on demographics, Cardiovascular disease (CVD) history,smoking habits, family history of CVD, alcohol consumption andmedication use prior to their first visit. The participants wereinvestigated for total cholesterol level, high-density lipoprotein (HDL)cholesterol, triglycerides, measured blood pressure, serum creatinineand diabetes mellitus. Diabetes was defined according to the guidelinesof the American Diabetes Association as a fasting plasma glucose ≧7.0mmol/l or the use of antidiabetic medication. Furthermore, informationon medication use was substantiated with use of pharmacy-dispensing datafrom all community pharmacies in the city of Groningen.

At baseline, CVD was defined by history of prior major cardiovascularevents (i.e., myocardial infarction, cerebrovascular accident), historyof coronary angioplasty and/or bypass surgery, ankle/brachial index lessthan 0.9, or presence of QS pattern based on the Minnesotaclassification (i.e., 1.1 and 1.2 codes). Individuals with CVD atpresentation were excluded from the study. Individuals with diabetes atpresentation were excluded from the study. Individuals having any cancerat presentation were excluded from the study. Individuals with a serumcreatinine >132 μmol/L were considered as having renal dysfunction andwere excluded from the study. The total number of individuals eligiblefor the study was n=5313. Thus, the population investigated in thisstudy was a representative population of apparently non-diseased people,representative for the general healthy population.

On the time point of inclusion, a blood sample was taken from eachindividual for making laboratory measurements (see below).

The investigated population was followed for ten years, and theoccurrence of various endpoints was recorded: The endpoints of the studywere incident cardiac events, incident cerebrovascular events,cardiovascular mortality, total cardiovascular events, incidentdiabetes, cancer mortality and total mortality. A cardiac event wasdefined as the sum of any of the following events: acute myocardialinfarction (ICD-code 410), acute and subacute ischemic heart disease(411), and the following procedures: coronary artery bypass grafting orpercutaneous transluminal coronary angioplasty. A cerebrovascular eventwas defined as the sum of any of the following events: occlusion orstenosis of the precerebral (433) or cerebral arteries (434).Cardiovascular mortality was defined as death due to a cardiac orcerebrovascular event. Total cardiovascular events were defined as thesum of cardiac events, cerebrovascular events and cardiovascularmortality. Diabetes was defined according to the guidelines of theAmerican Diabetes Association as a fasting plasma glucose ≧7.0 mmol/l orthe use of antidiabetic medication. Cancer mortality was defined asdeath due to cancer. Total mortality was defined as death of any cause.

Information (on hospitalization) for cardiovascular morbidity wasobtained from PRISMANT, the Dutch national registry of hospitaldischarge diagnoses. Data were coded according to the InternationalClassification of Diseases, 9th revision and the classification ofinterventions. In case a person had moved to an unknown destination, thedate on which the person was removed from the municipal registry wasused as censor date. Data on mortality were received from the municipalregister, and cause of death was obtained by linking the number of thedeath certificate to the primary cause of death as coded by the DutchCentral Bureau of Statistics.

Laboratory Assay

At baseline, blood samples were drawn after an overnight fast. Bloodsamples were drawn into chill tube containing EDTA. The samples wereimmediately centrifuged at 1500 g and 4° C. for 10 min, and thesupernatants stored in aliquots at −80° C. for further use. PCT wasmeasured as follows: A commercially available ultrasensitive sandwichassay for PCT was used (BRAHMS PCT LIA sensitive) (Morgenthaler et al.Clin Chem 2002; 48:788-90), which uses one antibody directed against thekatacalcin moiety of PCT as solid phase, and one antibody directedagainst the calcitonin moiety of PCT as labeled antibody (BRAHMS AG,Hennigsdorf, Germany). Recombinant PCT in various concentrations is usedas calibrator. A sample volume of 100 μl was used.

Statistical Analyses

Baseline descriptive statistics of the continuous variables werereported as mean±standard deviation (SD) and as median with 25th and75th percentiles, whenever the distribution of data was skewed. Thecategorical variables were presented as numbers and percentages. A χ2test was used to test the differences between groups for the categoricalvariables. The continuous data were compared by using Student's t testor a Mann-Whitney rank test if the variable was not normally distributedaccording to Kolmogorov-Smirnov test. To test the prediction value ofPCT for incidence events, we fitted separately Cox proportional hazardmodels with the development of the various events, which had beenrecorded, as the dependent variables. Because of a skewed distribution,logarithmic transformation (log 2) of PCT was applied in Cox regressionanalysis to compute hazard ratios (HR) and 95% confidence intervals(CIs) per doubling of PCT levels. P values of 0.05 or less fromtwo-sided tests were considered statistically significant. Additionally,hazard ratios were calculated for the second through the fifth PCTquintile in comparison to the first PCT quintile. All the statisticalanalyses were carried out via Statistical Package for Social Sciencesversion 16 (SPSS Inc, Chicago, Ill., USA) and S-PLUS software(Insightful Corp., Seattle, Wash., USA).

Results

The baseline characteristics of the study population are summarized inTable 1.

Table 2 shows the baseline PCT levels (all) and baseline PCT levels(≦0.1 ng/ml) stratified by gender.

Analyses were performed with samples containing PCT levels ≦0.1 ng/mland with samples containing PCT levels that were also higher than 0.1ng/ml. It was found that the prognostic value of PCT was essentially thesame for both of these sample.

PCT levels were significantly different between male and female (Table2) for the whole study group (P<0.001) as well as for subjects with PCTvalues ≦0.1 ng/ml (P<0.001).

Tables 3 and 4 show Hazard ratios, which describe the increase of riskfor developing an event, as indicated, per doubling of the PCTconcentration. For instance, a Hazard ratio of 2.23 for cardiovascularmortality means that an individual with a PCT concentration of, say,0.030 ng/mL has a 2.23-fold higher risk of dying from cardiovascularcomplications than an individual having a PCT concentration of 0.015ng/mL.

Table 3 shows Hazard Ratios (95% CI) for the prediction ofcardiovascular events as well as cancer, cardiovascular and totalmortality by PCT levels (all and ≦0.1 ng/ml, respectively) in the wholestudy group.

Table 4 shows Hazard Ratios (95% CI) for the prediction ofcardiovascular events as well as cancer, cardiovascular and totalmortality by PCT levels (all and ≦0.1 ng/ml, respectively) stratified bygender

Table 5 shows Hazard Ratios (95% CI) for the prediction of developmentof Type 2 Diabetes in the asymptomatic population by PCT levels (all and≦0.1 ng/ml).

Table 6 shows cut-off values and AUC ROC. From the entire investigatedpopulation PCT values >0.1 ng/mL were excluded from this analysis. Foreach type of event, the Area under the receiver operator characteristicscurve (AUC ROC) was calculated. Optimal cut-off values were defined asthe highest mathematical product of sensitivity and specificity, whichcould be reached for each type of event, when testing all possiblecut-off values. For the person skilled in the art it is clear that othercut-off values might be considered “optimal” depending on whethersensitivity, specificity, positive predictive value or negativepredictive value are important in the particular application.

Table 7 shows the adverse events according to PCT quintiles. From theentire investigated population PCT values >0.1 ng/mL were excluded fromthis analysis. The population was separated in PCT concentrationquintiles. For the various types of events the number of cases andproportion per quintile (in percent) are shown. The Hazard ratio fordevelopment of each type of event was calculated for quintiles Q2through Q5 by comparison with quintile Q1. The analysis for the eventtype diabetes is shown separately from the other events, because thenumber of persons, which could be followed for this event type wassmaller than for the other event types. The numbers of individuals perquintile are not perfectly even, because of a high frequency ofindividual values at those PCT concentrations separating the quintiles,which had to be attributed to either the upper or lower quintile at thisconcentration.

Taken together, the results show that elevated PCT levels inasymptomatic subjects are of prognostic value for predicting whether thesubject is at risk for suffering from an adverse event in the future.

Tables

TABLE 1 No. of participants 5313 Age- yr 47.3 ± 12   Men- no. (%) 2375(44.7) Body mass index- kg/m² 25.8 ± 4.1  Waist circumference- cm 86.9 ±12.7 Systolic blood pressure- mmHg 122.1 ± 18.2  Diastolic bloodpressure- mmHg 70.8 ± 9.4  Cholesterol- mmol/l 5.6 ± 1.1 HDLcholesterol- mmol/l 1.3 ± 0.4

TABLE 2 PCT levels (all) at baseline in ng/ml (n = 5313) Men (n = 2353)Women (n = 2960) P value* 0.018 (0.015-0.022) 0.014 (0.012-0.017)<0.0001 PCT levels (≦0.1 ng/ml) at baseline in ng/ml (n = 5292) Men (n =2337) Women (n = 2955) P value* 0.018 (0.015-0.022) 0.014 (0.012-0.017)<0.0001 *Calculated using Mann-Whitney U test

TABLE 3 PCT (all) at baseline (n = 5313) Cerebrovascular CardiovascularTotal Cancer Total Cardiac events events mortality Cardiovascularmortality mortality (n = 98) (n = 61) (n = 30) events (n = 172) (n = 91)(n = 148) 1.53 1.50 1.52 1.52 1.53 1.55 (1.26-1.87) (1.15-1.96)(1.04-2.20) (1.31-1.77) (1.24-1.89) (1.32-1.81) PCT (≦0.1 ng/ml) atbaseline (n = 5292) Cerebrovascular Cardiovascular Total Cancer TotalCardiac events events mortality Cardiovascular mortality mortality (n =98) (n = 61) (n = 30) events (n = 172) (n = 89) (n = 146) 2.33 2.18 2.232.78 1.79 2.07 (1.65-3.30) (1.40-3.40) (1.18-4.18) (1.75-2.96)(1.20-2.65) (1.55-2.78)

TABLE 4 PCT (all) in men at baseline (n = 2353) Cardiovascular TotalCancer Total Cardiac events Cerebrovascular mortality Cardiovascularmortality mortality (n = 46) events (n = 30) (n = 17) events (n = 85) (n= 41) (n = 73) 1.51 1.41 0.91 1.40 1.95 1.75 (1.01-2.26) (0.82-2.42)(0.33-2.46) (1.02-1.93) (1.44-2.66) (1.34-2.29) PCT (all) in women atbaseline (n = 2960) Cardiovascular Total Cancer Total Cardiac eventsCerebrovascular mortality Cardiovascular mortality mortality (n = 52)events (n = 31) (n = 13) events (n = 87) (n = 50) (n = 75) 1.55 1.531.67 1.56 1.28 1.43 (1.23-1.94) (1.13-2.09) (1.17-2.37) (1.31-1.85)(0.84-1.93) (1.12-1.83) PCT (≦0.1 ng/ml) in men at baseline (n = 2337)Cardiovascular Total Cancer Total Cardiac events Cerebrovascularmortality Cardiovascular mortality mortality (n = 46) events (n = 30) (n= 17) events (n = 85) (n = 40) (n = 72) 2.04 1.80 1.20 1.79 2.29 2.08(1.18-3.50) (0.90-3.60) (1.12-1.29) (1.18-2.71) (1.31-4.01 (1.35-3.19)PCT (≦0.1 ng/ml) in women at baseline (n = 2950) Cardiovascular TotalCancer Total Cardiac events Cerebrovascular mortality Cardiovascularmortality mortality (n = 52) events (n = 31) (n = 13) events (n = 87) (n= 49) (n = 74) 2.93 2.67 1.14 2.98 1.54 2.12 (1.81-4.72) (1.44-4.97)(1.07-1.21) (2.06-4.29) (0.83-2.85) (1.36-3.30)

TABLE 5 PCT (all) at baseline All participants Men Women (123/4206)(62/1870) (61/2336) 1.78 (1.44-2.20) 1.61 (1.13-2.30) 2.18 (1.48-3.21)PCT (≦0.1 ng/ml) at baseline All participants Men Women (121/4193)(60/1859) (61/2334) 2.11 (1.51-2.95) 1.43 (0.84-2.44) 3.25 (2.00-5.30)

TABLE 6 Optimal PCT cut-off Area under the receiver concentrationoperator characteristics Type of event [ng/mL] curve (AUC ROC) Cardiacevents 0.0155 0.634 Cerebrovascular events 0.0215 0.628 Cardiovascularmortality 0.0215 0.640 Total cardiovascular 0.0155 0.635 events Cancermortality 0.0155 0.585 Total mortality 0.0205 0.610 Diabetes 0.01850.627

TABLE 7 Q1 Q2 Q3 Q4 Q5 No. of subjects 1045    1069     958    1214    1006     Procalcitonin 0.007-0.012 0.013-0.014 0.015-0.016 0.017-0.0200.021-0.098 Range [ng/mL] Total mortality No. of cases (%) 12 (1.1) 28(2.6) 23 (2.4) 34 (2.8) 49 (4.9) <0.001 Hazard Ratio 1.0 2.32 2.12 2.474.35 (95% CI) (1.18-4.57) (1.06-4.27) (1.28-4.78) (2.32-8.19) Totalcardiovascular mortality No. of cases (%)  1 (0.1)  5 (0.5)  7 (0.7)  6(0.5) 11 (1.1) 0.007 Hazard Ratio 1.0 4.98 7.76 5.25 11.75  (95% CI) (0.58-42.62)  (0.95-63.11)  (0.63-43.58)  (1.52-91.04) Cancer mortalityNo. of cases (%) 11 (1.1) 15 (1.4) 14 (1.5) 23 (1.9) 26 (2.6) 0.004Hazard Ratio 1.0 1.36 1.41 1.83 2.52 (95% CI) (0.62-2.96) (0.64-3.11)(0.89-3.75) (1.25-5.11) Cardiac events No. of cases (%)  8 (0.8) 12(1.1) 20 (2.1) 24 (2.0) 34 (3.4) <0.001 Hazard Ratio 1.0 1.50 2.79 2.654.60 (95% CI) (0.61-3.68) (1.23-6.34) (1.19-5.90) (2.13-9.93)Cerebrovascular events No. of cases (%)  6 (0.6) 10 (0.9)  8 (0.8) 14(1.2) 23 (2.3) 0.001 Hazard Ratio 1.0 1.66 1.48 2.05 4.13 (95% CI)(0.60-4.57) (0.51-4.27) (0.79-5.32)  (1.68-10.15) Total cardiovascularevents No. of cases (%) 13 (1.2) 25 (2.3) 33 (3.4) 40 (3.3) 61 (6.1)<0.001 Hazard Ratio 1.0 1.93 2.83 2.72 5.10 (95% CI) (0.98-3.76)(1.49-5.38) (1.45-5.08) (2.80-9.29) No. of persons 845    834    745   978    791    Procalcitonin 0.007-0.012 0.013-0.014 0.015-0.0160.017-0.020 0.021-0.096 Range [ng/mL] Diabetes No. of cases (%) 13 (1.5)16 (1.9) 21 (2.8) 27 (2.8) 44 (5.6) <0.001 Odds Ratio 1.0 1.25 1.86 1.823.77 (95% CI) (0.60-2.62) (0.92-3.73) (0.93-3.54) (2.01-7.05)

1-17. (canceled)
 18. A method for determining an increased risk of anapparently healthy subject, preferably a human, for suffering from anadverse event, comprising: providing a sample from said subject;determining the level of procalcitonin (PCT) or of a fragment thereof insaid sample; and determining whether said subject has an increased riskfor suffering from an adverse event based on said PCT level, whereinsaid adverse event is not an event caused by an acute exogene inducedadverse event and/or exogene induced trauma and wherein the followingsubjects are excluded from the group of apparently healthy subjects:neonates between 1 day to 3 days and patients after surgery wherein thesurgery has been conducted within the last 7 days and post-traumapatients and persons which experienced said trauma within the last 7days.
 19. The method of claim 18, wherein PCT has the amino acidsequence shown as SEQ ID NO.
 1. 20. The method of claim 18, wherein saidPCT fragment comprises (contains) at least 12 amino acids, preferablymore than 50 amino acids, most preferably more than 100 amino acids, orthe amino acids 2 to 116 or 3 to 116 of the amino acid sequence shown asSEQ ID NO.
 1. 21. The method of claim 18, wherein said leveldetermination is performed using a diagnostic assay, preferably animmunoassay.
 22. The method of claim 18, wherein the adverse event ischosen from the group consisting of cardiac events, cardiovascularevents, cerebrovascular events, cardiovascular mortality, diabetes,cancer mortality, and death.
 23. The method of claim 18, wherein therisk determination is performed by comparing the determined PCT or PCTfragment level with the median of the level of PCT or fragments thereofin an ensemble of predetermined samples from a population of apparentlyhealthy subjects, or comparing the determined PCT or PCT fragment levelwith a quantile of the level of PCT or fragments thereof in an ensembleof predetermined samples from a population of apparently healthysubjects, or performing calculations based on Cox Proportional Hazardsanalysis or on risk index calculations such as the Net ReclassificationIndex (NRI) or the Integrated Discrimination Index (IDI).
 24. The methodof claim 18, wherein the subject is free of symptomatic infections. (PCTlevel not increased above normal range)
 25. The method of claim 18,wherein the sample is a bodily fluid, in particular blood, serum,plasma, cerebrospinal fluid, urine, saliva, sputum, or a pleuraleffusion.
 26. The method of claim 18, wherein said subject is determinedof being at risk for suffering from an adverse event when said PCT levelin a serum or plasma sample is at least 0.015 ng/ml, preferably at least0.02 ng/ml.
 27. The method of claim 18, wherein said subject isdetermined of being at an increased risk for suffering from a cardiacevent or a cardiovascular event when said PCT level in a serum sample isat least 0.0155 ng/ml.
 28. The method of claim 18, wherein said subjectis determined of being at an increased risk for suffering from acerebrovascular event or from dying due to a cardiovascular cause whensaid PCT level in a serum sample is at least 0.0215 ng/ml.
 29. Themethod of claim 18, wherein said subject is determined of being at anincreased risk for dying from cancer when said PCT level in a serumsample is at least 0.0155 ng/ml.
 30. The method of claim 18, whereinsaid subject is determined of being at an increased risk for dying whensaid PCT level in a serum sample is at least 0.0205 ng/ml.
 31. Themethod of claim 18, wherein said subject is determined of being at anincreased risk for suffering from diabetes when said PCT level in aserum sample is at least 0.0185 ng/ml.
 32. The method of claim 18,further comprising determining a clinical parameter that is selectedfrom the group consisting of gender, age, systolic blood pressure,systolic blood pressure, diastolic blood pressure, body mass index,waist circumference, waist-hip ratio, and smoking habit; or a laboratoryparameter that is selected from the group consisting of fasting blood orplasma glucose concentration, triglycerides, cholesterol concentration,and HDL cholesterol concentration and subfractions thereof, LDLcholesterol concentration and subfractions thereof, cystatin C, insulin,CRP, natriuretic peptides of the A- and B-type as well as theirprecursors and fragments thereof including ANP, proANP, NT-proANP,MR-proANP, BNP, proBNP, NT-proBNP, GDF15, ST2, procalcitonin andfragments thereof, pro-adrenomedullin and fragments thereof includingADM, PAMP, MR-proADM, CT-proADM, pro-Endothelin-1 and fragments thereofincluding CT-proET-1, NT-proET-1, big-Endothelin-1, and Endothelin-1; ora genetic parameter that is selected from the group consisting of singlenucleotide polymorphisms (SNP), and mutations, in particular related todiabetes heredity.
 33. The method of claim 18, further comprising takingat least one preventive measure for avoiding said adverse event fromoccurring in said subject.
 34. Use of a method of claim 18 fordetermining an increased risk of an apparently healthy subject forsuffering from an adverse event.